N-phenylacryloyl amino acid / peptide with oxidation resistance as well as preparation method and application of N-phenylacryloyl amino acid / peptide

Abstract

The invention discloses an N-phenylacryloyl amino acid / peptide with oxidation resistance as well as a preparation method and application of the N-phenylacryloyl amino acid / peptide. The antioxidant capacity of amino acid and peptide is improved on the basis of N-phenylacryloyl modification, N-phenylacryloyl amino acid or N-phenylacryloyl peptide compounds with remarkable antioxidant capacity are synthesized by a chemical method or an enzyme method, and the antioxidant activity of the N-phenylacryloyl amino acid or N-phenylacryloyl peptide compounds is evaluated by adopting a DPPH clearance rate experiment. The N-phenylacryloylated amino acid and the N-phenylacryloylated peptide can effectively prolong the shelf life and maintain nutritional ingredients in a food system, and can be used for resisting skin oxidation and delaying skin aging in the field of cosmetics. Experiments show that the compounds can play a remarkable anti-oxidation effect under the condition of low addition amount. The invention provides a safe and efficient natural anti-oxidation solution for food and cosmetic industries, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method for enhancing the antioxidant capacity of amino acids and peptides based on N-phenylpropenylation modification and its application in the food and cosmetic fields. Background Technology

[0002] Currently, the safety and quality stability of food and cosmetics are receiving much attention, and oxidation is one of the key factors leading to a decline in food quality and skin aging.

[0003] In the food industry, oxidation not only causes the deterioration of components such as fats and pigments, resulting in unpleasant rancidity, foul odors, and discoloration, but also destroys nutrients such as vitamins and unsaturated fatty acids, reducing the nutritional value of food. Traditional synthetic antioxidants, such as BHA (tert-butylhydroxyanisole) and BHT (di-tert-butyl-p-methylphenol), while possessing good antioxidant effects, have seen increasing scrutiny regarding their safety in recent years. Long-term intake may pose potential health risks, such as inducing cell mutations and affecting liver and kidney function. Therefore, finding safe and effective natural antioxidants has become a pressing issue for the food industry. Simultaneously, with the increasing consumer demand for natural and healthy foods, developing antioxidants derived from natural plants and microorganisms to replace traditional synthetic antioxidants has become a research hotspot in the field of food preservation and quality improvement.

[0004] In the cosmetics industry, oxidative damage to the skin is one of the main causes of skin aging, pigmentation, inflammation, and other problems. Environmental factors such as ultraviolet radiation, pollutants, and free radicals can induce the skin to produce excessive reactive oxygen species (ROS), damaging the skin cells' antioxidant defense system and triggering lipid peroxidation, protein oxidation, and DNA damage within skin cells. This accelerates the skin aging process, causing the skin to lose elasticity and radiance, resulting in wrinkles, sagging, and dullness. Therefore, the cosmetics industry has been committed to developing highly effective antioxidants to combat oxidative damage to the skin, delay skin aging, and meet people's needs for beauty and skincare. However, existing antioxidants still have shortcomings in terms of stability and persistence, failing to fully exert their antioxidant effects.

[0005] Amino acids and peptides, as essential components of living organisms, possess a variety of physiological functions and are widely available and highly safe. Recent studies have revealed that amino acids and peptides exhibit certain antioxidant capabilities, exerting their antioxidant effects through mechanisms such as chelating metal ions, scavenging free radicals, and reducing oxidized substances. However, their inherent antioxidant capacity is relatively limited, making it difficult to achieve ideal antioxidant effects in practical applications, thus restricting their widespread use in the food and cosmetic industries. Therefore, effectively enhancing the antioxidant capacity of amino acids and peptides has become crucial for promoting their application.

[0006] The literature Son, S., & Lewis, BA (2002). Free radical scavenging and antioxidant activity of caffeic acid amide and ester analogues: Structure-activity relationship. Journal of Agricultural and Food Chemistry, 50(3), 468-472. discloses a potential antioxidant. This literature mainly studies the synthesized caffeic acid amide and ester analogues as potential antioxidants. However, the research in this literature is limited to amide and ester analogues of caffeic acid and does not involve other phenylacrylic acids. In addition, its synthesis method is relatively complex and not applicable to the food and cosmetic industries. Furthermore, the lipophilic antioxidant in the literature shows good antioxidant activity in linoleic acid emulsion systems; however, its application in aqueous environments may be limited.

[0007] Based on this research background, this invention significantly enhances the antioxidant properties of amino acids and peptides by cleverly modifying them with N-phenylpropenylation, opening up new avenues for the research and development of antioxidants in food and cosmetics, and is expected to meet the market's urgent demand for safe, efficient, and natural antioxidants. Summary of the Invention

[0008] Based on the above, the primary objective of this invention is to modify the structure of amino acids and peptides using N-phenylacrylylation. Specifically, by introducing phenylacrylyl groups such as cinnamyl, p-coumaryl, caffeoyl, ferulicyl, or sinapicyl into the amino position of amino acids and peptides, a series of modified products with unique structures are formed. These modified compounds exhibit significantly enhanced antioxidant capacity due to the conjugated system of the phenylacrylyl group and its tendency to release hydrogen atoms.

[0009] Another object of the present invention is to provide a method for evaluating the antioxidant capacity of the N-phenylacryloyl amino acid compound.

[0010] The objective of this invention is achieved through the following technical solution:

[0011] An N-phenylacryloyl amino acid / peptide with antioxidant capabilities, comprising N-phenylacryloyl amino acid and N-phenylacryloyl peptide.

[0012] This invention provides a method for preparing N-phenylacryloyl amino acids / peptides, comprising one of the following methods:

[0013] Method (1): A chemical synthesis method for the antioxidant compound N-phenylacryloyl amino acid, comprising the following steps:

[0014] Preparation of phenylacryloyl chloride: Phenylic acid carboxylic acid was dissolved in dry DCM, then oxaloyl chloride was added, DMF was added dropwise as a catalyst, the reaction mixture was stirred at room temperature, the solvent was evaporated, and the product was thoroughly dried in vacuum to obtain phenylacryloyl chloride;

[0015] Preparation of N-phenylacryloyl amino acids / peptides: Phenylacetyl chloride was dissolved in diethyl ether, and amino acids or peptides were dissolved in NaOH solution. The solutions were combined and stirred at room temperature. Water and diethyl ether were added to the reaction mixture, and the two phases were stirred and separated. The aqueous phase was adjusted to acidity and extracted with dichloromethane. The solvent was dried and evaporated to obtain N-phenylacryloyl amino acids / peptides.

[0016] The molar ratio of the phenylacrylic acid, oxaloyl chloride, and DCM is (5-15):(6-18):(0.3-1); the molar ratio of the phenylacryloyl chloride, amino acid or peptide, diethyl ether, and water is (5-15):(5-15):(0.1-0.5):(1-5); and the concentration of the NaOH solution is 0.1-2 mol / L.

[0017] Method (2): An enzymatic synthesis method for an antioxidant compound N-phenylacryloyl amino acid / peptide, comprising the following steps:

[0018] N-phenylacryloyl carboxylic acids and amino acids or peptides are dissolved in water, the pH is adjusted, and the reaction is carried out under the catalysis of food-grade or cosmetic-grade enzymes. After the reaction is completed, the enzyme is inactivated, cooled to room temperature, and extracted with diethyl ether. The aqueous phase is separated and the pH is adjusted to acidic. The mixture is then extracted with dichloromethane, and the solvent is dried and evaporated to obtain N-phenylacryloyl amino acid / peptide.

[0019] The molar ratio of the phenylacrylic acid, amino acid or peptide, and water is (5-15):(0.5-1.5):(1-5); the enzymatic hydrolysis pH is 6.0-8.0; the food-grade enzyme includes any one or combination of glutaminase, trypsin, alkaline protease, neutral protease, and lipase; the dosage of the food-grade enzyme is 0.1%-2.0% w / v; the enzymatic hydrolysis temperature is 25-50℃, and the enzymatic hydrolysis time is 6-48 h; the enzyme inactivation temperature is 80-100℃, and the time is 15-25 min.

[0020] Furthermore, the phenyl acrylic acid carboxylic acids described in this invention include, but are not limited to: 3-phenyl-2-acrylic acid, 3-(2-hydroxyphenyl)-2-acrylic acid, 3-(3-hydroxyphenyl)-2-acrylic acid, 3-(4-hydroxyphenyl)-2-acrylic acid, 3-(2-methoxyphenyl)-2-acrylic acid, 3-(3-methoxyphenyl)-2-acrylic acid, 3-(4-methoxyphenyl)-2-acrylic acid, 3-(3,4-methoxyphenyl)-2-acrylic acid, 3-(3,4-dihydroxyphenyl)-2-acrylic acid, 3-(4-hydroxy-3-methoxyphenyl)-2-acrylic acid, and 3-(4-hydroxy-3,5-dimethoxyphenyl)-2-acrylic acid.

[0021] Furthermore, the amino acids described in this invention include, but are not limited to, any one of: alanine, cysteine, aspartic acid, phenylalanine, glutamic acid, histidine, isoleucine, lysine, leucine, methionine, asparagine, glutamine, arginine, serine, threonine, valine, tryptophan, tyrosine, proline, glycine, ornithine, theanine, sarcosine, γ-aminobutyric acid, or β-alanine.

[0022] Furthermore, the peptides described in this invention include, but are not limited to, peptide segments composed of two or more of the above-mentioned amino acids.

[0023] The antioxidant compound N-phenylacryloyl amino acid or peptide obtained by the above preparation method exhibits excellent antioxidant activity, especially significant DPPH scavenging activity, with an IC50 value of [missing information]. 50 The value is in the range of 0.01 to 15 mg / mL.

[0024] The antioxidant compound N-phenylacryloyl amino acid with significant DPPH scavenging activity obtained by the above preparation method has the following general structural formula:

[0025]

[0026] The N-phenylpropenylated peptide, an antioxidant compound with significant DPPH scavenging activity, obtained by the above preparation method, has the following general structural formula:

[0027]

[0028] in

[0029] R1, R2, R3, and R4 represent hydrogen, hydroxyl, or methoxy groups, respectively, independently of each other.

[0030] R5 represents an amino acid residue, preferably a protein amino acid, ornithine, theanine, sarcosine, γ-aminobutyric acid or β-alanine residue.

[0031] R6 represents a peptide chain residue, preferably a residue of a peptide that has antioxidant capacity, such as carnosine, glutathione, or alanyl-histyl-proline.

[0032] This invention also provides an antioxidant evaluation method for the above-mentioned N-phenylacryloyl amino acid / peptide. The antioxidant evaluation methods include determination of DPPH free radical scavenging capacity, determination of ABTS free radical scavenging capacity, and determination of FRAP total antioxidant capacity.

[0033] This invention discloses an N-phenylacryloyl-L-amino acid / peptide with antioxidant capabilities, which can be synthesized through both chemical and enzymatic methods. The N-phenylacryloyl amino acid / peptide is obtained by acylation of a phenylacrylic acid compound with an amino acid or peptide. The chemical method is an optimization of traditional synthesis methods, employing a two-phase reaction system of ether and water to achieve highly efficient and selective synthesis of the target product, reduce by-product formation, simplify subsequent separation and purification steps, and improve product purity. Compared to the chemical method, the enzymatic method utilizes specific enzyme-catalyzed reactions, exhibiting advantages of high selectivity and mild conditions, making it more suitable for food systems. This technology solves the problems of low efficiency, poor selectivity, and easy generation of by-products in traditional synthesis methods, offering advantages of high yield, high purity, and environmental friendliness.

[0034] The present invention also provides the application of the above-mentioned N-phenylacryloyl amino acid / peptide with antioxidant capacity in food or cosmetics, wherein the amount of N-phenylacryloyl amino acid / peptide added is 0.001% to 0.5% of the weight of the food or cosmetic system.

[0035] This invention enhances the antioxidant capacity of amino acids and peptides through N-phenylacrylylation modification. N-phenylacrylamide amino acids or N-phenylacrylated peptide compounds with significant antioxidant capacity were synthesized using chemical or enzymatic methods, and their antioxidant activity was evaluated using a DPPH scavenging assay. Experimental results show that the N-phenylacrylamide amino acids or peptides prepared in this invention exhibit excellent antioxidant effects even at low concentrations, with an IC50 value of [missing information]. 50 The concentration ranges from 0.01 to 15 mg / mL. In the food industry, the compounds of this invention can serve as natural antioxidants, effectively extending the shelf life of food and preventing quality degradation caused by oxidation, such as inhibiting rancidity and maintaining food color and nutritional components. In the cosmetics industry, they can combat skin oxidation, slow down the skin aging process, improve dull skin, and enhance the skin's antioxidant defense capabilities. They can be used to prepare various skincare products with antioxidant and anti-aging effects, such as serums, creams, and lotions.

[0036] The present invention has the following advantages and beneficial effects compared with the prior art:

[0037] (1) The present invention utilizes N-phenylacrylylation reaction to modify the structure of amino acids and peptides, introducing phenylacrylyl groups into the amino positions of amino acids and peptides to form a series of modified products with phenylacrylyl groups substituted for amino groups.

[0038] (2) The present invention uses N-phenylpropenyl acylation modification process, which is green and safe. The raw materials are conventional chemical reagents or enzymes that meet food safety standards. There is no potential risk to human health, and it can effectively improve the antioxidant properties of amino acids and peptides.

[0039] (3) The N-phenylacryloyl amino acid and peptide compounds successfully developed in this invention have excellent antioxidant properties and can be used as natural antioxidants in the food and cosmetic fields, with broad application prospects.

[0040] (4) The preparation process of the present invention is simple and easy to implement, and the cost is low. It is convenient for large-scale industrial production and helps to promote the rapid development of related industries.

[0041] (5) The N-feruloylphenylalanine prepared in this invention exhibits significant antioxidant capacity at concentrations of 0.01–0.1 mg / mL, and its DPPH free radical scavenging capacity has an IC50 value of [missing value]. 50 The value was 0.046 mg / mL, which is similar to the antioxidant properties (IC50) of the traditional antioxidant vitamin E. 50 The concentration of 0.043 mg / mL indicates that the compound of this invention can exert an antioxidant effect comparable to vitamin E at extremely low concentrations, making it a potential high-efficiency natural antioxidant that can be widely used in the food and cosmetic fields. Attached Figure Description

[0042] Figure 1 The structure of N-cinnamyltryptophan synthesized by chemical method in Example 1 is shown below.

[0043] Figure 2 This is a flowchart of the chemical synthesis of N-p-coumaroyltyrosine in Example 2.

[0044] Figure 3 The structure of N-feruloylphenylalanine synthesized by chemical method in Example 3 is shown below.

[0045] Figure 4 The structure of N-coumaroyl carnosine synthesized by chemical method in Example 4 is shown below.

[0046] Figure 5 The N-cinnamylphenylalanine synthesized by enzymatic method in Example 5 13 C spectrum (A) and 1 H NMR spectrum (B).

[0047] Figure 6The structure of N-coumaroyltryptophan synthesized by enzymatic method in Example 6 is shown.

[0048] Figure 7 The structure of N-feruloyl glutathione synthesized by enzymatic method in Example 7 is shown below.

[0049] Figure 8 The DPPH scavenging curve of vitamin E was determined in Example 9.

[0050] Figure 9 The DPPH scavenging curves for tryptophan (A), N-cinnamoyltryptophan (B), and cinnamic acid (C) as determined in Example 10 are shown.

[0051] Figure 10 The DPPH scavenging curves for p-coumaric acid (A), phenylalanine (B), and N-p-coumaryl phenylalanine (C) determined in Example 11 are shown.

[0052] Figure 11 The DPPH scavenging curve of N-coumaroyltryptophan was determined in Example 12.

[0053] Figure 12 The DPPH scavenging curve of N-coumaroyltyrosine was determined in Example 13.

[0054] Figure 13 The DPPH scavenging curves of ferulic acid (A) and N-feruloylphenylalanine (B) as determined in Example 14 are shown.

[0055] Figure 14 The values ​​are the peroxide value and anisidine value of soybean oil with added N-coumaroyl carnosine, as determined in Example 15. Detailed Implementation

[0056] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included within the scope of the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those skilled in the art can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of the present invention to realize and apply the technology of the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0057] Example 1: Chemical Synthesis of N-Cinnamyl Tryptophan

[0058] Preparation of cinnamoyl chloride: Dissolve 10 mmol of cinnamic acid in 40 mL of dry DCM, then add 12 mmol of oxaloyl chloride and 2-3 drops of DMF as a catalyst. Stir the reaction mixture overnight at room temperature. Evaporate the solvent and dry the product thoroughly under vacuum to obtain cinnamoyl chloride. The product can be used without further purification.

[0059] Preparation of N-cinnamoyltryptophan: 10 mmol of cinnamoyl chloride was dissolved in 20 mL of diethyl ether, and 10 mmol of tryptophan was dissolved in 40 mL of 1 M NaOH solution. The solutions were combined and stirred overnight at room temperature. The next day, 40 mL of water and 20 mL of diethyl ether were added to the reaction mixture, and the mixture was stirred and the two phases were separated. The aqueous phase was adjusted to pH 2-3, and the product was extracted with dichloromethane. The solvent was dried and evaporated to obtain N-cinnamoyltryptophan.

[0060] Figure 1 The structure of N-cinnamyltryptophan synthesized by chemical method in Example 1 is shown below.

[0061] Example 2 Chemical Synthesis of N-p-Coumaroyl Tyrosine

[0062] Acetylation protection: In an ice bath, 10 mmol of p-coumaric acid, 64 mmol of sodium hydroxide, and 20 mL of water were mixed and stirred until cinnamic acid was completely dissolved. 26 mmol of acetic anhydride was slowly added dropwise, controlling the dropping rate to maintain the reaction temperature below 10°C. After the addition was complete, the mixture was allowed to warm naturally to room temperature, and the reaction was continued with stirring for 2 hours. After the reaction was complete, the pH of the solution was adjusted to 2.0 with concentrated hydrochloric acid, and a white solid precipitated. The solid was filtered under reduced pressure, the filter cake was washed twice with water, recrystallized from anhydrous ethanol, and dried to obtain acetylated p-coumaric acid.

[0063] Preparation of acetyl-p-coumaryl chloride: 15 mmol of acetyl-p-coumaryl chloride was dissolved in 40 mL of dry DCM, then 18 mmol of oxaloyl chloride was added, and 2-3 drops of DMF were added dropwise as a catalyst. The reaction mixture was stirred overnight at room temperature. The solvent was evaporated, and the product was thoroughly dried under vacuum to obtain acetyl-p-coumaryl chloride. The product can be used without further purification.

[0064] Preparation of N-p-coumaryl tyrosine: 15 mmol of acetyl-p-coumaryl chloride was dissolved in 30 mL of diethyl ether, and 15 mmol of tyrosine was dissolved in 60 mL of 1 M NaOH solution. The solutions were combined and stirred overnight at room temperature. The next day, 40 mmol of solid sodium hydroxide was added to the reaction mixture, and the mixture was heated to 30 °C to hydrolyze the acetyl groups for 2 hours. After the reaction was complete, 40 mL of water and 20 mL of diethyl ether were added, the mixture was stirred, and the two phases were separated. The aqueous phase was adjusted to pH 2-3, and the product was extracted with dichloromethane. The solvent was dried and evaporated to obtain N-p-coumaryl tyrosine.

[0065] Figure 2This is a flowchart of the chemical synthesis of N-p-coumaroyltyrosine in Example 2.

[0066] Example 3 Chemical Synthesis of N-feruloylphenylalanine

[0067] Acetylation protection: In an ice bath, mix 5 mmol ferulic acid, 32 mmol sodium hydroxide, and 10 mL water, stirring until cinnamic acid is completely dissolved. Slowly add 13 mmol acetic anhydride, controlling the adding rate to maintain the reaction temperature below 10°C. After the addition is complete, allow the mixture to warm naturally to room temperature and continue stirring for 2 hours. After the reaction is complete, adjust the pH of the solution to 2.0 with concentrated hydrochloric acid, precipitating a white solid. Filter under reduced pressure, wash the filter cake twice with water, recrystallize with anhydrous ethanol, and dry to obtain acetylated ferulic acid.

[0068] Preparation of acetylferuloyl chloride: Dissolve 5 mmol of acetyl-p-coumaryl chloride in 20 mL of dry DCM, then add 6 mmol of oxaloyl chloride and 2-3 drops of DMF as a catalyst. Stir the reaction mixture overnight at room temperature. Evaporate the solvent and dry the product thoroughly under vacuum to obtain acetylferuloyl chloride. The product can be used without further purification.

[0069] Preparation of N-feruloylphenylalanine: 5 mmol of acetyl-p-coumaryl chloride was dissolved in 10 mL of diethyl ether, and 5 mmol of phenylalanine was dissolved in 20 mL of 1 M NaOH solution. The solutions were combined and stirred overnight at room temperature. The next day, 20 mmol of solid sodium hydroxide was added to the reaction mixture, and the mixture was heated to 30 °C to hydrolyze the acetyl group for 2 hours. After the reaction was complete, 40 mL of water and 20 mL of diethyl ether were added, the mixture was stirred, and the two phases were separated. The aqueous phase was adjusted to pH 2-3, and the product was extracted with dichloromethane. The solvent was dried and evaporated to obtain N-feruloylphenylalanine.

[0070] Figure 3 The structure of N-feruloylphenylalanine synthesized by chemical method in Example 3 is shown below.

[0071] Example 4 Chemical Synthesis of N-p-Coumaroyl Carnosine

[0072] Acetylation protection: In an ice bath, 5 mmol of p-coumaric acid, 32 mmol of sodium hydroxide, and 10 mL of water were mixed and stirred until cinnamic acid was completely dissolved. 13 mmol of acetic anhydride was slowly added dropwise, controlling the dropping rate to maintain the reaction temperature below 10°C. After the addition was complete, the mixture was allowed to warm naturally to room temperature, and the reaction was continued with stirring for 2 hours. After the reaction was complete, the pH of the solution was adjusted to 2.0 with concentrated hydrochloric acid, and a white solid precipitated. The solid was filtered under reduced pressure, the filter cake was washed twice with water, recrystallized from anhydrous ethanol, and dried to obtain acetylated p-coumaric acid.

[0073] Preparation of acetylated p-coumaryl chloride: 5 mmol of acetylated p-coumaric acid was dissolved in 20 mL of dry DCM, then 6 mmol of oxaloyl chloride was added, along with 2-3 drops of DMF as a catalyst. The reaction mixture was stirred overnight at room temperature. The solvent was evaporated, and the product was thoroughly dried under vacuum to obtain acetylated p-coumaryl chloride. The product can be used without further purification.

[0074] Preparation of N-p-coumaryl carnosine: 5 mmol of acetyl-p-coumaryl chloride was dissolved in 10 mL of diethyl ether, and 5 mmol of carnosine was dissolved in 20 mL of 1 M NaOH solution. The solutions were combined and stirred overnight at room temperature. The next day, 10 mmol of solid sodium hydroxide was added to the reaction mixture, and the mixture was heated to 30 °C to hydrolyze the acetyl groups for 2 hours. After the reaction was complete, 40 mL of water and 20 mL of diethyl ether were added, the mixture was stirred, and the two phases were separated. The aqueous phase was adjusted to pH 2-3, and the product was extracted with dichloromethane. The solvent was dried and evaporated to obtain N-p-coumaryl carnosine.

[0075] Figure 4 The structure of N-coumaroyl carnosine synthesized by chemical method in Example 4 is shown below.

[0076] Example 5 Enzymatic preparation of N-cinnamylphenylalanine

[0077] 10 mmol of cinnamic acid and 1 mmol of phenylalanine were dissolved in 25 mL of water, and the pH was adjusted to 7.0. The reaction was carried out under the catalysis of food-grade enzyme Sumizyme FP-G (enzyme dosage: 1.0 w / v%) at 37 °C for 24 h. After the reaction, the enzyme was inactivated at 90 °C for 15 min, cooled to room temperature, and extracted with diethyl ether. The aqueous phase was separated and the pH was adjusted to 2-3. The product was extracted with dichloromethane. The solvent was dried and evaporated to obtain N-cinnamylphenylalanine.

[0078] HPLC detection conditions: The mobile phases were 0.1% (v / v) trifluoroacetic acid-acetonitrile solution (A) and 0.1% (v / v) trifluoroacetic acid-aqueous solution (B), respectively. A gradient elution method was used, with the following elution program: 0 min: 15% A + 85% B; 25 min: 35% A + 65% B; 30 min: 35% A + 65% B; 35 min: 15% A + 85% B; elution ended. Detection was performed using an LC-UV100 ultraviolet detector (Wufeng, Guangzhou). The chromatographic column was a Shodex C18-120-5 4E, with a length of 250 mm and an inner diameter of 4.6 mm. The detection wavelength was 220 nm, the column temperature was set to 30 °C, the injection volume was 10 μL, and the flow rate was 1.0 mL / min.

[0079] NMR characterization: The prepared N-cinnamylphenylalanine was dissolved in tritium dimethyl sulfoxide (DMSO-d6). A comprehensive NMR characterization was performed using a Bruker AVANCE III HD 600MHz spectrometer. 1 H and 13 C10 nuclear magnetic resonance (NMR) spectroscopy analysis.

[0080] The elution times of N-cinnamoylphenylalanine, cinnamic acid, and phenylalanine were compared by HPLC. The structure of purified cinnamoylphenylalanine was analyzed by NMR.

[0081] Nuclear magnetic resonance spectroscopy confirmed the structure of N-cinnamylphenylalanine. Figure 5 A and B respectively show the N-cinnamoylphenylalanine 1 H and 13 C NMR spectrum. 1H NMR (600MHz, DMSO) δ7.57-7.51(m, 2H), 7.42-7.35(m, 4H), 7.28(dd, J=14.5, 4.4Hz, 5H), 6.74(d , J=15.8Hz, 1H), 4.65-4.52 (m, 1H), 3.13 (dd, J=13.8, 4.7Hz, 1H), 2.94 (dd, J=13.9, 9.5Hz, 1H). 13C NMR (151MHz, DMSO) δ 173.00, 164.90, 139.14, 137.70, 134.79, 129.12, 128.95, 128.40, 128.21, 127.57, 126.44, 121.78, 53.78, 36.83. Proton NMR ( 1 H NMR spectrum Figure 5 A) was obtained in dimethyl sulfoxide (DMSO) at 600 MHz, showing characteristic peaks at δ7.57–7.51 (m, 2H), δ7.42–7.35 (m, 4H), and δ7.28 (dd, J = 14.5, 4.4 Hz, 5H), indicating the aromatic protons of the cinnamoyl group. A distinct doublet at δ6.74 (d, J = 15.8 Hz, 1H) corresponds to the protons on the benzene ring of phenylalanine. Aliphatic protons, which can be attributed to the α- and β-protons of the phenylalanine side chain, were observed in the regions of δ4.65–4.52 (m, 1H), δ3.13 (dd, J = 13.8, 4.7 Hz, 1H), and δ2.94 (dd, J = 13.9, 9.5 Hz, 1H). Carbon-13 NMR records in DMSO at 151 MHz (…) 13 C NMR spectrum Figure 5B) confirmed the presence of carbonyl carbons at δ173.00 and δ164.90, and aromatic carbons in the range of δ139.14 to δ121.78. Aliphatic carbons of phenylalanine residues were observed at δ53.78 and δ36.83. These spectroscopic data collectively validate the successful synthesis and structural integrity of N-cinnamylphenylalanine.

[0082] Example 6 Enzymatic preparation of N-p-coumaroyltryptophan

[0083] 15 mmol of phenylpropionic acid carboxylic acid and 1.5 mmol of tryptophan were dissolved in 30 mL of water, and the pH was adjusted to 8.0. The reaction was carried out under the catalysis of food-grade enzyme Sumizyme DPP-G (enzyme dosage: 1.5 w / v%) at 30 °C for 18 h. After the reaction, the enzyme was inactivated at 80 °C for 20 min, cooled to room temperature, and extracted with diethyl ether. The aqueous phase was separated and the pH was adjusted to 2-3. The product was extracted with dichloromethane. The solvent was dried and evaporated to obtain N-coumaroyl tryptophan.

[0084] Figure 6 The structure of N-coumaroyltryptophan synthesized by enzymatic method in Example 6 is shown.

[0085] Example 7 Enzymatic Synthesis of N-Ferulayl Glutathione

[0086] 5 mmol ferulic acid and 0.5 mmol glutathione were dissolved in 20 mL of water, and the pH was adjusted to 6.0. The reaction was carried out under the catalysis of cosmetic-grade collagenase (enzyme addition: 2 w / v%) at 25 °C for 30 h. After the reaction, the enzyme was inactivated at 100 °C for 10 min, cooled to room temperature, and extracted with diethyl ether. The aqueous phase was separated and the pH was adjusted to 2-3. The product was extracted with dichloromethane. The solvent was dried and evaporated to obtain N-feruloyl glutathione.

[0087] Figure 7 The structure of N-feruloyl glutathione synthesized by enzymatic method in Example 7 is shown below.

[0088] Example 8: DPPH free radical scavenging ability test

[0089] 1.0.12 mg / mL DPPH ethanol solution: Accurately weigh 12 mg of 1,1-diphenyl-2-trinitrophenylhydrazine, dilute to 100 mL with 95% ethanol, dissolve by sonication, and store at low temperature in the dark.

[0090] Sample solution preparation: Prepare test sample solutions of different concentrations (0.001-0.1 mg / mL). Water-soluble samples are diluted with water to prepare multi-concentration samples, and oil-soluble samples are diluted with 95% ethanol to prepare multi-concentration samples.

[0091] 2. Reaction system preparation: Add 1 mL of sample solution to a 10 mL test tube, add 2 mL of 95% ethanol and 1 mL of DPPH ethanol solution, mix well, and repeat 6 times in parallel. Simultaneously set up a sample control group: replace the DPPH ethanol solution with 95% ethanol solution; a blank group: replace the sample solution with 95% ethanol solution or aqueous solution (sample solvent); and a blank control group: replace the sample solution with 95% ethanol solution or aqueous solution (sample solvent) and simultaneously replace the DPPH ethanol solution with 95% ethanol solution.

[0092] 3. Reaction conditions: React the mixed solution at room temperature for 15 minutes in the dark.

[0093] 4. Measurement: Transfer the reacted solution into a 1cm cuvette and measure the absorbance at 517nm using a UV-Vis spectrophotometer. The DPPH free radical scavenging rate is calculated according to formula (1):

[0094]

[0095] In the formula:

[0096] T—Absorbance of the sample group, i.e., the absorbance of the solution after the sample reacts with DPPH;

[0097] T0—Absorbance value of the sample control group;

[0098] C—Absorbance of the blank group, i.e., absorbance of the DPPH solution without sample;

[0099] C0—Absorbance value of the blank control group.

[0100] Example 9: DPPH free radical scavenging ability test of vitamin E (VE)

[0101] 1.0.12 mg / mL DPPH ethanol solution: Accurately weigh 12 mg of 1,1-diphenyl-2-trinitrophenylhydrazine, dilute to 100 mL with 95% ethanol, dissolve by sonication, and store at low temperature in the dark.

[0102] Sample solution preparation: Prepare vitamin E solutions of various concentrations (0.01-0.1 mg / mL) using 95% ethanol.

[0103] 2. Preparation of the reaction system: Take 1 mL of V E Add the solution to a 10 mL test tube, add 2 mL of 95% ethanol and 1 mL of DPPH ethanol solution, mix well, and repeat 6 times in parallel. Simultaneously set up a sample control group: replacing the DPPH ethanol solution with 95% ethanol solution; a blank group: replacing the sample solution with 95% ethanol solution; and a blank control group: replacing both the sample solution and DPPH ethanol solution with 95% ethanol solution.

[0104] 3. Reaction conditions: React the mixed solution at room temperature for 15 minutes in the dark.

[0105] 4. Measurement: Transfer the reacted solution into a 1 cm cuvette and measure the absorbance at 517 nm using a UV-Vis spectrophotometer. Calculate the DPPH radical scavenging rate according to formula (1).

[0106] from Figure 8 As can be seen, the horizontal axis represents the concentration of vitamin E (mg / mL), and the vertical axis represents the DPPH free radical scavenging rate (%). A straight line fits between the vitamin E concentration and the DPPH free radical scavenging rate, with the equation y = 1198.9x - 2.1454, where R0... 2 =0.9992, indicating a good linear relationship between vitamin E concentration and DPPH free radical scavenging rate. This shows that within the tested concentration range, the DPPH free radical scavenging rate increases linearly with increasing vitamin E concentration. This linear relationship allows for the prediction of DPPH free radical scavenging rate of vitamin E at different concentrations using a linear regression equation. The IC50 of vitamin E's DPPH free radical scavenging rate was then calculated. 50 The value (the test concentration of the sample corresponding to achieving 50% scavenging effect) is 0.0435 mg / mL, which meets the IC50 value of the positive control vitamin E in the T / SHRH 006-2018 Cosmetics - Free Radical (DPPH) Scavenging Test Method Standard. 50 The value should be in the range of 0.02 mg / mL to 0.06 mg / mL, indicating that the DPPH free radical scavenging capacity testing system in Example 8 is effective. Vitamin E can be used as a benchmark for measuring antioxidant capacity; when comparing different antioxidants, the lower the IC50 value, the stronger the antioxidant capacity of the substance.

[0107] Example 10: DPPH free radical scavenging capacity test of cinnamic acid, tryptophan, and N-cinnamoyltryptophan

[0108] The DPPH free radical scavenging capacity of cinnamic acid, tryptophan, and N-cinnamoyltryptophan was determined according to the DPPH free radical scavenging capacity test in Example 8.

[0109] The test results are shown in Table 1 and Figure 9 As shown. From Figure 9 As can be seen from A, the DPPH free radical scavenging rate of tryptophan gradually increases with increasing concentration. The linear regression equation is y = 2.1079x + 7.8975, and the correlation coefficient R0 is [value missing]. 2 =0.9528, indicating that tryptophan has a certain scavenging ability against DPPH free radicals within the tested concentration range, but the overall effect is relatively limited. Figure 9B showed that the DPPH radical scavenging rate of N-cinnamoyl tryptophan significantly increased with increasing concentration. The linear regression equation was y = 3.6256x + 19.122, and the correlation coefficient R0 was [value missing]. 2 =0.9667, indicating that N-cinnamoyl tryptophan has a stronger DPPH free radical scavenging ability and exhibits significant antioxidant effects even at low concentrations. Unlike tryptophan and N-cinnamoyl tryptophan, such as Figure 9 As shown in Figure C, cinnamic acid did not exhibit significant DPPH free radical scavenging ability within the tested concentration range (1-10 mg / mL). This indicates that cinnamic acid, when used alone, has weak antioxidant capacity or may have no antioxidant activity.

[0110] DPPH radical scavenging ability tests showed that cinnamylation significantly enhanced the antioxidant capacity of tryptophan, while cinnamic acid alone had no significant antioxidant effect. N-cinnamoyltryptophan exhibited a stronger DPPH radical scavenging effect than tryptophan even at low concentrations, with its DPPH radical scavenging ability IC50 value being [missing information]. 50 The value decreased from 19.97 mg / mL to 8.51 mg / mL, demonstrating a significant antioxidant effect.

[0111] Example 11: DPPH radical scavenging ability test of p-coumaric acid, phenylalanine and N-p-coumarylphenylalanine

[0112] The DPPH free radical scavenging abilities of p-coumaric acid, phenylalanine, and N-p-coumaryl phenylalanine were determined according to the DPPH free radical scavenging ability test in Example 8.

[0113] The test results are shown in Table 1 and Figure 10 As shown. From Figure 10 As can be seen from A, p-coumaric acid has a certain scavenging ability against DPPH free radicals. Within the tested concentration range, the DPPH free radical scavenging rate of p-coumaric acid gradually increases with increasing concentration. The linear regression equation is y = 2.3236x + 15.976, and the correlation coefficient R is [missing value]. 2 =0.9644, calculate its IC 50 The value was 14.64 mg / mL. Figure 10 The B-value shows that phenylalanine did not exhibit significant DPPH free radical scavenging ability, indicating that when used alone, phenylalanine has weak antioxidant capacity or may have no antioxidant activity. Figure 10 The C-values ​​show that phenylalanine exhibits a certain DPPH radical scavenging ability after coumarinylation modification. The linear regression equation for the DPPH radical scavenging rate of N-coumarin is y = 2.736x + 9.8566, with a correlation coefficient of R0. 2 =0.9942, IC 50The value was 14.67 mg / mL. Experiments showed that the antioxidant effect of N-p-coumaroylphenylalanine was comparable to that of p-coumaric acid, indicating that p-coumarylation modification can impart antioxidant activity to amino acids that originally lacked it.

[0114] Example 12 Test of DPPH free radical scavenging ability of N-p-coumaryl tryptophan

[0115] The DPPH free radical scavenging ability of N-coumaryl tryptophan was determined according to the DPPH free radical scavenging ability test in Example 8.

[0116] The test results are shown in Table 1 and Figure 11 As shown in the figure, the DPPH radical scavenging rate of N-p-coumaryl tryptophan significantly increases with increasing concentration. The linear regression equation is y = 6.0134x + 16.593, and the correlation coefficient R0 is [value missing]. 2 =0.9887, indicating that N-coumaroyl tryptophan possesses a strong DPPH radical scavenging ability. The DPPH radical scavenging ability test shows that coumarylation modification significantly enhances the antioxidant capacity of tryptophan. N-coumaroyl tryptophan exhibits a strong DPPH radical scavenging effect even at low concentrations, indicating that coumarylation modification is an effective strategy to enhance the antioxidant properties of amino acids, especially those with inherent antioxidant activity, providing a scientific basis for the development of novel natural antioxidants.

[0117] Example 13: DPPH free radical scavenging ability test of tyrosine and N-p-coumaryl tyrosine

[0118] The DPPH free radical scavenging ability of N-p-coumaroyltyrosine was determined according to the DPPH free radical scavenging ability test in Example 8.

[0119] The test results are shown in Table 1 and Figure 12 As shown. Due to the solubility limitation of tyrosine, its DPPH radical scavenging rate is low at low concentrations, and it is difficult to prepare high-concentration solutions for testing. The measured DPPH radical scavenging rate of N-p-coumaryltyrosine increased significantly with increasing concentration, and the linear regression equation was y = 2.7619x + 14.727, with a correlation coefficient R. 2 =0.9818. Its IC 50 The value was 12.77 mg / mL. This indicates that coumarin acylation significantly enhanced the antioxidant capacity of tyrosine, making the originally poorly water-soluble tyrosine exhibit a stronger DPPH free radical scavenging effect in aqueous solution. This suggests that phenylacrylic acid acylation modification can not only enhance the antioxidant properties of amino acids but also improve their water solubility, thereby increasing their application potential as novel natural antioxidants in the food and cosmetic fields.

[0120] Example 14: DPPH free radical scavenging ability test of ferulic acid and N-feruloylphenylalanine

[0121] The DPPH free radical scavenging capacity of ferulic acid and N-feruloylphenylalanine was determined according to the DPPH free radical scavenging capacity test in Example 8.

[0122] The test results are shown in Table 1 and Figure 13 As shown. From Figure 13 As can be seen from A, ferulic acid possesses excellent DPPH radical scavenging ability, and the DPPH radical scavenging rate gradually increases with increasing concentration. The linear regression equation is y = 1287.9x + 9.2326, and the correlation coefficient R0 is [value missing]. 2 =0.9939, its IC 50 The value was 0.03 mg / mL, indicating that ferulic acid has a good scavenging ability against DPPH free radicals within the tested concentration range. Figure 13 B showed that ferulic acid acylation significantly enhanced the antioxidant capacity of phenylalanine, and the DPPH radical scavenging rate of N-feruloylphenylalanine increased significantly with increasing concentration. The linear regression equation was y = 1008.9x + 3.5034, and the correlation coefficient R0 was [value missing]. 2 =0.9993, its IC 50 The value was 0.05 mg / mL, and the antioxidant capacity was close to that of vitamin E, indicating that ferulic acid acylation modification is an effective strategy that can significantly enhance the antioxidant properties of amino acids. This provides a scientific basis for the development of novel natural and highly efficient antioxidants and further expands its application prospects as a novel natural antioxidant in the food and cosmetic fields.

[0123] Table 1 Evaluation of the antioxidant capacity of N-phenylpropenylated amino acids

[0124]

[0125] Note: "—" indicates that DPPH free radical scavenging ability was not measured.

[0126] Example 15 Antioxidant Application Test in Food Systems

[0127] 1. Sample preparation: Fresh soybean oil was selected as the test sample, and different concentrations of N-p-coumaroyl carnosine (0.001%-0.1%, w / w) were added. At the same time, a blank group (no antioxidant added) and a positive control group (0.1% carnosine added) were set up.

[0128] 2. Accelerated oxidation experiment: Vegetable oil with added antioxidants was placed in a constant temperature environment of 60℃, and samples were taken after 15 days to determine its peroxide value and anisidine value.

[0129] 3. Results: The test results are as follows Figure 14 As shown, compared with vegetable oils without added antioxidants, the vegetable oils with added N-p-coumaroyl carnosine showed a significantly slower increase in peroxide value and anisidine value over the same time period, indicating that the compounds of this invention can effectively inhibit the oxidation process of vegetable oils and extend their shelf life. Compared with carnosine, N-p-coumaroyl carnosine exhibited better antioxidant effects at the same concentration, proving the effectiveness of N-phenylacrylic acid acylation modification in enhancing antioxidant capacity. This further demonstrates the effectiveness of phenylacrylic acid acylation modification in enhancing the antioxidant capacity of peptides, providing the food industry with a novel and highly efficient natural antioxidant option.

[0130] Example 16: Antioxidant Application Test of Cosmetic System

[0131] 1. Sample preparation: Emulsions were prepared according to conventional cosmetic formulations, and different concentrations of N-feruloyl glutathione (0.01%-0.5%, w / w) were added. A blank group (without any antioxidants) and a positive control group (with 0.5% glutathione added) were also set up.

[0132] 2. Stability test: The prepared emulsion was placed in a constant temperature environment of 40℃, and its appearance, viscosity and antioxidant activity were observed periodically.

[0133] 3. Antioxidant activity test: The DPPH free radical scavenging rate of each sample at different time points was determined according to the method in Example 8.

[0134] 3. Results: The test results are shown in Table 2. After being stored at 40℃ for 3 months, the emulsion containing N-feruloyl glutathione maintained a high DPPH free radical scavenging rate, significantly higher than that of the blank group and the positive control group. This indicates that N-phenylacryloyl amino acid / peptide has good antioxidant effects in cosmetic systems. Furthermore, the emulsion containing N-feruloyl glutathione showed no significant changes in appearance or viscosity during the test period, and no layering or discoloration occurred. In contrast, the blank group emulsion showed slight layering, darkening in color, and a slight decrease in viscosity after 3 months of storage, indicating that the compounds of this invention have good stability in cosmetic systems. Through antioxidant application tests in cosmetic systems, N-phenylacryloyl amino acid / peptide exhibited significant antioxidant properties and stability. They can effectively resist oxidation reactions in cosmetic systems, extend the shelf life of products, and maintain the appearance and efficacy of products. This provides the cosmetic industry with a novel and highly efficient natural antioxidant option with broad application prospects.

[0135] Table 2. Antioxidant capacity, viscosity, and appearance of N-feruloyl glutathione in cosmetic emulsions.

[0136]

Claims

1. An N-phenylacryloyl amino acid / peptide with antioxidant capacity, characterized in that, It includes N-phenylacryloyl amino acids and N-phenylacryloyl peptides, wherein the general structural formula of the N-phenylacryloyl amino acid is as follows: The general structural formula of the N-phenylpropenylated peptide is as follows: in R1, R2, R3, and R4 represent hydrogen, hydroxyl, or methoxy groups, respectively, independently of each other. R5 represents an amino acid residue; R6 represents a peptide chain residue.

2. The N-phenylacryloyl amino acid / peptide with antioxidant capacity according to claim 1, characterized in that, The amino acids include any one of alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, tyrosine, serine, threonine, cysteine, methionine, aspartic acid, glutamic acid, asparagine, glutamine, histidine, arginine, lysine, glycine, alanine, cysteine, aspartic acid, phenylalanine, glutamic acid, histidine, isoleucine, lysine, leucine, methionine, asparagine, glutamine, arginine, serine, threonine, valine, tryptophan, tyrosine, proline, glycine, ornithine, theanine, sarcosine, γ-aminobutyric acid, or β-alanine.

3. The N-phenylacryloyl amino acid / peptide with antioxidant capacity according to claim 1, characterized in that, The peptide is a peptide segment composed of two or more of the above-mentioned amino acids.

4. The method for preparing an N-phenylacryloyl amino acid / peptide with antioxidant capacity as described in claim 1, characterized in that, This includes chemical synthesis and enzymatic synthesis methods, which involve acylation of phenylacrylic acid compounds with amino acids or peptides to obtain N-phenylacryloyl amino acids / peptides.

5. The preparation method according to claim 4, characterized in that, The chemical synthesis method includes the following steps: Preparation of phenylacryloyl chloride: Phenylic acid carboxylic acid was dissolved in dry DCM, then oxaloyl chloride was added, DMF was added dropwise as a catalyst, the reaction mixture was stirred at room temperature, the solvent was evaporated, and the product was thoroughly dried in vacuum to obtain phenylacryloyl chloride; Preparation of N-phenylacryloyl amino acids / peptides: Phenylacetyl chloride was dissolved in diethyl ether, and amino acids or peptides were dissolved in NaOH solution. The solutions were combined and stirred at room temperature. Water and diethyl ether were added to the reaction mixture, and the two phases were stirred and separated. The aqueous phase was adjusted to acidity and extracted with dichloromethane. The solvent was dried and evaporated to obtain N-phenylacryloyl amino acids / peptides.

6. The preparation method according to claim 4, characterized in that, The enzymatic synthesis method includes the following steps: N-phenylacryloyl carboxylic acids and amino acids or peptides are dissolved in water, the pH is adjusted, and the reaction is carried out under the catalysis of food-grade or cosmetic-grade enzymes. After the reaction is completed, the enzyme is inactivated, cooled to room temperature, and extracted with diethyl ether. The aqueous phase is separated and the pH is adjusted to acidic. The mixture is then extracted with dichloromethane, and the solvent is dried and evaporated to obtain N-phenylacryloyl amino acid / peptide.

7. The preparation method according to claim 5, characterized in that, The molar ratio of the phenylacrylic acid, oxaloyl chloride, and DCM is (5-15):(6-18):(0.3-1); the molar ratio of the phenylacryloyl chloride, amino acid or peptide, diethyl ether, and water is (5-15):(5-15):(0.1-0.5):(1-5); and the concentration of the NaOH solution is 0.1-2 mol / L.

8. The preparation method according to claim 6, characterized in that, The molar ratio of the phenylacrylic acid, amino acid or peptide, and water is (5-15):(0.5-1.5):(1-5); the enzymatic hydrolysis pH is 6.0-8.0; the food-grade enzyme includes any one or combination of glutaminase, trypsin, alkaline protease, neutral protease, and lipase; the dosage of the food-grade enzyme is 0.1%-2.0% w / v; the enzymatic hydrolysis temperature is 25-50℃, and the enzymatic hydrolysis time is 6-48 h; the enzyme inactivation temperature is 80-100℃, and the time is 15-25 min.

9. The application of the N-phenylacryloyl amino acid / peptide with antioxidant capacity as described in claim 1 in food, characterized in that, The amount of N-phenylacryloyl amino acid / peptide added is 0.001% to 0.5% of the weight of the food system.

10. The application of the N-phenylacryloyl amino acid / peptide with antioxidant capacity as described in claim 1 in cosmetics, characterized in that, The amount of N-phenylacryloyl amino acid / peptide added is 0.001% to 0.5% of the weight of the cosmetic system.

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